Serine/Threonine Phosphorylation of ShcA
2002; Elsevier BV; Volume: 277; Issue: 33 Linguagem: Inglês
10.1074/jbc.m203229200
ISSN1083-351X
AutoresAmir Faisal, Mahmoud El‐Shemerly, Daniel Heß, Yoshikuni Nagamine,
Tópico(s)Ubiquitin and proteasome pathways
ResumoSerine phosphorylation of the ShcA signaling molecule has been reported recently. In this work, we have identified 12-O-tetradecanoylphorbol-13-acetate (TPA)- and growth factor-induced serine/threonine phosphorylation sites in p52Shc and p66Shc. Among them, Ser29 in p52Shc (equivalent to Ser138 in p66Shc) was phosphorylated only after TPA stimulation. Phosphorylation of this site together with the intact phosphotyrosine-binding domain was essential for ShcA binding to the protein-tyrosine phosphatase PTP-PEST. TPA-induced ShcA phosphorylation at this site (and hence, its association with PTP-PEST) was inhibited by a protein kinase C-specific inhibitor and was induced by overexpression of constitutively active mutants of protein kinase Cα, -ε, and -δ isoforms. Insulin also induced ShcA/PTP-PEST association, although to a lesser extent than TPA. Overexpression of a PTP-PEST binding-defective mutant of p52Shc (S29A) enhanced insulin-induced ERK activation in insulin receptor-overexpressing HIRc-B cells. Consistent with this, p52Shc S29A was more tyrosine-phosphorylated than wild-type p52Shc after insulin stimulation. Thus, we have identified a new mechanism whereby serine phosphorylation of ShcA controls the ability of its phosphotyrosine-binding domain to bind PTP-PEST, which is responsible for the dephosphorylation and down-regulation of ShcA after insulin stimulation. Serine phosphorylation of the ShcA signaling molecule has been reported recently. In this work, we have identified 12-O-tetradecanoylphorbol-13-acetate (TPA)- and growth factor-induced serine/threonine phosphorylation sites in p52Shc and p66Shc. Among them, Ser29 in p52Shc (equivalent to Ser138 in p66Shc) was phosphorylated only after TPA stimulation. Phosphorylation of this site together with the intact phosphotyrosine-binding domain was essential for ShcA binding to the protein-tyrosine phosphatase PTP-PEST. TPA-induced ShcA phosphorylation at this site (and hence, its association with PTP-PEST) was inhibited by a protein kinase C-specific inhibitor and was induced by overexpression of constitutively active mutants of protein kinase Cα, -ε, and -δ isoforms. Insulin also induced ShcA/PTP-PEST association, although to a lesser extent than TPA. Overexpression of a PTP-PEST binding-defective mutant of p52Shc (S29A) enhanced insulin-induced ERK activation in insulin receptor-overexpressing HIRc-B cells. Consistent with this, p52Shc S29A was more tyrosine-phosphorylated than wild-type p52Shc after insulin stimulation. Thus, we have identified a new mechanism whereby serine phosphorylation of ShcA controls the ability of its phosphotyrosine-binding domain to bind PTP-PEST, which is responsible for the dephosphorylation and down-regulation of ShcA after insulin stimulation. mitogen-activated protein kinase phosphotyrosine-binding collagen homologous region 1 Src homology 2 epidermal growth factor 12-O-tetradecanoylphorbol-13-acetate, ERK, extracellular signal-regulated kinase protein kinase C protein-tyrosine phosphatase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase hemagglutinin Dulbecco's modified Eagle's medium calf serum fibroblast growth factor high pressure liquid chromatography Protein phosphorylation, a key component in the regulation of signaling pathways controlling many fundamental physiological processes, is determined not only by protein kinases, but also by protein phosphatases (1Hunter T. Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2577) Google Scholar). One of the pathways controlled by protein phosphorylation and dephosphorylation is the mitogen-activated protein kinase (MAPK)1 pathway that converts the receptor signals into a variety of outputs (1Hunter T. Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2577) Google Scholar, 2Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4223) Google Scholar). Shc adapter/docking proteins are an important component of this pathway because they are involved in transducing the activation signals from receptor or cytoplasmic tyrosine kinases to downstream signaling cascades (3Pelicci G. Lanfrancone L. Salcini A.E. Romano A. Mele S. Grazia Borrello M. Segatto O., Di Fiore P.P. Pelicci P.G. Oncogene. 1995; 11: 899-907PubMed Google Scholar, 4Bonfini L. Migliaccio E. Pelicci G. Lanfrancone L. Pelicci P.G. Trends Biochem. Sci. 1996; 21: 257-261Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 5Baldari C.T. Pelicci G., Di Somma M.M. Milia E. Giuli S. Pelicci P.G. Telford J.L. Oncogene. 1995; 10: 1141-1147PubMed Google Scholar). At least three genes, shcA, shcB, and shcC, are known to encode Shc proteins. These share an amino-terminal phosphotyrosine-binding (PTB) domain, a central proline/glycine-rich region (CH1), and a carboxyl-terminal Src homology 2 (SH2) domain (6Nakamura T. Sanokawa R. Sasaki Y. Ayusawa D. Oishi M. Mori N. Oncogene. 1996; 13: 1111-1121PubMed Google Scholar, 7O'Bryan J.P. Songyang Z. Cantley L. Der C.J. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2729-2734Crossref PubMed Scopus (95) Google Scholar, 8Pelicci G. Lanfrancone L. Grignani F. McGlade J. Cavallo F. Forni G. Nicoletti I. Pawson T. Pelicci P.G. Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1131) Google Scholar, 9Pelicci G. Dente L., De Giuseppe A. Verducci-Galletti B. Giuli S. Mele S. Vetriani C. Giorgio M. Pandolfi P.P. Cesareni G. Pelicci P.G. Oncogene. 1996; 13: 633-641PubMed Google Scholar). ShcA exists in three isoforms in mammalian cells, p46, p52, and p66, which differ only in the extent of their amino-terminal sequence and are produced through alternative splicing and differential use of translation initiation sites (10Luzi L. Confalonieri S., Di Fiore P.P. Pelicci P.G. Curr. Opin. Genet. Dev. 2000; 10: 668-674Crossref PubMed Scopus (188) Google Scholar).ShcA has two modules of phosphotyrosine recognition with different specificities, an amino-terminal PTB domain and a carboxyl-terminal SH2 domain (11van der Geer P. Wiley S. Lai V.K. Olivier J.P. Gish G.D. Stephens R. Kaplan D. Shoelson S. Pawson T. Curr. Biol. 1995; 5: 404-412Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Thus, it can associate with tyrosine-phosphorylated proteins such as receptor tyrosine kinases and cytoplasmic proteins (e.g. SHIP2) (12Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Crossref PubMed Scopus (561) Google Scholar). ShcA has been found to be phosphorylated rapidly and efficiently by all tyrosine kinases tested to date (10Luzi L. Confalonieri S., Di Fiore P.P. Pelicci P.G. Curr. Opin. Genet. Dev. 2000; 10: 668-674Crossref PubMed Scopus (188) Google Scholar). These phosphorylation sites have been mapped to Tyr239, Tyr240, and Tyr317 in the CH1 domain (13Salcini A.E. McGlade J. Pelicci G. Nicoletti I. Pawson T. Pelicci P.G. Oncogene. 1994; 9: 2827-2836PubMed Google Scholar, 14Gotoh N. Tojo A. Shibuya M. EMBO J. 1996; 15: 6197-6204Crossref PubMed Scopus (115) Google Scholar, 15van der Geer P. Wiley S. Gish G.D. Pawson T. Curr. Biol. 1996; 6: 1435-1444Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). These phosphorylated tyrosines then serve as binding sites for the SH2 domain of adapter protein Grb2 (15van der Geer P. Wiley S. Gish G.D. Pawson T. Curr. Biol. 1996; 6: 1435-1444Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 16Rozakis-Adcock M. McGlade J. Mbamalu G. Pelicci G. Daly R., Li, W. Batzer A. Thomas S. Brugge J. Pelicci P.G. Schlessinger J. Pawson T. Nature. 1992; 360: 689-692Crossref PubMed Scopus (825) Google Scholar, 17Songyang Z. Shoelson S.E. McGlade J. Olivier P. Pawson T. Bustelo X.R. Barbacid M. Sabe H. Hanafusa H., Yi, T. et al.Mol. Cell. Biol. 1994; 14: 2777-2785Crossref PubMed Scopus (829) Google Scholar), which is constitutively associated with SOS, a ubiquitously expressed Ras guanine nucleotide exchange factor. This binding leads to recruitment of SOS to the plasma membrane, an event considered sufficient to induce Ras activation. Consistent with this model, overexpression of ShcA can transform mouse fibroblasts and induce differentiation of PC12 cells (8Pelicci G. Lanfrancone L. Grignani F. McGlade J. Cavallo F. Forni G. Nicoletti I. Pawson T. Pelicci P.G. Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1131) Google Scholar).In addition to tyrosine phosphorylation, p66Shc can also be phosphorylated at serine/threonine residues in response to epidermal growth factor (EGF) (18Okada S. Kao A.W. Ceresa B.P. Blaikie P. Margolis B. Pessin J.E. J. Biol. Chem. 1997; 272: 28042-28049Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), 12-O-tetradecanoylphorbol-13-acetate (TPA) (19El-Shemerly M.Y. Besser D. Nagasawa M. Nagamine Y. J. Biol. Chem. 1997; 272: 30599-30602Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), UV stress (20Migliaccio E. Giorgio M. Mele S. Pelicci G. Reboldi P. Pandolfi P.P. Lanfrancone L. Pelicci P.G. Nature. 1999; 402: 309-313Crossref PubMed Scopus (1460) Google Scholar), Taxol (21Yang C.P. Horwitz S.B. Cancer Res. 2000; 60: 5171-5178PubMed Google Scholar), and endothelin-1 (22Foschi M. Franchi F. Han J., La Villa G. Sorokin A. J. Biol. Chem. 2001; 276: 26640-26647Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). EGF-induced serine/threonine phosphorylation of p66Shc has been implicated in the negative regulation of the MAPK pathway (18Okada S. Kao A.W. Ceresa B.P. Blaikie P. Margolis B. Pessin J.E. J. Biol. Chem. 1997; 272: 28042-28049Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). In contrast, it has been suggested that the TPA-induced serine/threonine phosphorylation of p52Shc/p66Shc is involved in ERK activation because it leads to an increase in ShcA/Grb2 association without an increase in ShcA tyrosine phosphorylation (19El-Shemerly M.Y. Besser D. Nagasawa M. Nagamine Y. J. Biol. Chem. 1997; 272: 30599-30602Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Similarly, a protein kinase C (PKC)-dependent increase in Shc/Grb2 association after TPA treatment in MCF7 cells has been attributed to ERK activation (23Sato H. Ogata H. De Luca L.M. Oncogene. 2000; 19: 2904-2912Crossref PubMed Scopus (27) Google Scholar). Finally, Migliaccio et al. (20Migliaccio E. Giorgio M. Mele S. Pelicci G. Reboldi P. Pandolfi P.P. Lanfrancone L. Pelicci P.G. Nature. 1999; 402: 309-313Crossref PubMed Scopus (1460) Google Scholar) reported that serine phosphorylation of p66Shc regulates stress-induced apoptotic response and life span in mammals.It has been shown that p52Shc/p66Shc proteins are associated with the protein-tyrosine phosphatase PTP-PEST and that this association can be enhanced in HeLa cells by PKC activators like TPA, but not by EGF (24Habib T. Herrera R. Decker S.J. J. Biol. Chem. 1994; 269: 25243-25246Abstract Full Text PDF PubMed Google Scholar). The sites involved in the association of p52ShcA/p66ShcA and PTP-PEST were mapped to the PTB domain in ShcA and an NPLH sequence in the carboxyl terminus of PTP-PEST (25Charest A. Wagner J. Jacob S. McGlade C.J. Tremblay M.L. J. Biol. Chem. 1996; 271: 8424-8429Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). This was the first report showing phosphotyrosine-independent binding of the PTB domain of ShcA to a target protein. Murine PTP-PEST is a ubiquitously expressed cytosolic phosphatase of 112 kDa characterized by the presence of a so-called PEST sequence that is thought to confer protein stability (26Charest A. Wagner J. Shen S.H. Tremblay M.L. Biochem. J. 1995; 308: 425-432Crossref PubMed Scopus (62) Google Scholar). PTP-PEST is phosphorylated by PKC and protein kinase A at Ser39 and Ser435, and this serine phosphorylation down-regulates its activity (27Garton A.J. Tonks N.K. EMBO J. 1994; 13: 3763-3771Crossref PubMed Scopus (106) Google Scholar). Association with other proteins (28Mauro L.J. Dixon J.E. Trends Biochem. Sci. 1994; 19: 151-155Abstract Full Text PDF PubMed Scopus (180) Google Scholar) may also control the activity of protein-tyrosine phosphatases, and ShcA has been suggested to recruit PTP-PEST to its substrates for dephosphorylation (24Habib T. Herrera R. Decker S.J. J. Biol. Chem. 1994; 269: 25243-25246Abstract Full Text PDF PubMed Google Scholar). PTP-PEST has been shown recently to associate with and dephosphorylate ShcA in B cells, contributing to negative regulation of lymphocyte activation via inactivation of the Ras pathway (29Davidson D. Veillette A. EMBO J. 2001; 20: 3414-3426Crossref PubMed Scopus (100) Google Scholar). Activated insulin receptor phosphorylates various cellular substrates at tyrosine residues (30White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar). ShcA is one of these substrates and has been shown to play an important role in insulin-induced ERK activation (31Yamauchi K. Pessin J.E. J. Biol. Chem. 1994; 269: 31107-31114Abstract Full Text PDF PubMed Google Scholar, 32Sasaoka T. Draznin B. Leitner J.W. Langlois W.J. Olefsky J.M. J. Biol. Chem. 1994; 269: 10734-10738Abstract Full Text PDF PubMed Google Scholar, 33Ishihara H. Sasaoka T. Ishiki M. Takata Y. Imamura T. Usui I. Langlois W.J. Sawa T. Kobayashi M. J. Biol. Chem. 1997; 272: 9581-9586Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Tyrosine phosphorylation of ShcA at residue 317 has been shown to play an important role in signal transduction to MAPK by insulin in Rat1 fibroblasts expressing the insulin receptor (33Ishihara H. Sasaoka T. Ishiki M. Takata Y. Imamura T. Usui I. Langlois W.J. Sawa T. Kobayashi M. J. Biol. Chem. 1997; 272: 9581-9586Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar).In this report, we have identified TPA- and growth factor-induced serine/threonine phosphorylation sites in ShcA. One of these phosphorylation sites has been characterized and shown to be involved in binding to the protein-tyrosine phosphatase PTP-PEST. Phosphorylation of this site was induced by overexpression of some constitutively active isoforms of PKC, but not by others. Furthermore, binding of PTP-PEST to Shc down-regulated insulin-induced ERK activation. We have shown also that TPA-induced ERK activation does not involve serine phosphorylation of Shc in mouse embryo fibroblasts. Protein phosphorylation, a key component in the regulation of signaling pathways controlling many fundamental physiological processes, is determined not only by protein kinases, but also by protein phosphatases (1Hunter T. Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2577) Google Scholar). One of the pathways controlled by protein phosphorylation and dephosphorylation is the mitogen-activated protein kinase (MAPK)1 pathway that converts the receptor signals into a variety of outputs (1Hunter T. Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2577) Google Scholar, 2Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4223) Google Scholar). Shc adapter/docking proteins are an important component of this pathway because they are involved in transducing the activation signals from receptor or cytoplasmic tyrosine kinases to downstream signaling cascades (3Pelicci G. Lanfrancone L. Salcini A.E. Romano A. Mele S. Grazia Borrello M. Segatto O., Di Fiore P.P. Pelicci P.G. Oncogene. 1995; 11: 899-907PubMed Google Scholar, 4Bonfini L. Migliaccio E. Pelicci G. Lanfrancone L. Pelicci P.G. Trends Biochem. Sci. 1996; 21: 257-261Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 5Baldari C.T. Pelicci G., Di Somma M.M. Milia E. Giuli S. Pelicci P.G. Telford J.L. Oncogene. 1995; 10: 1141-1147PubMed Google Scholar). At least three genes, shcA, shcB, and shcC, are known to encode Shc proteins. These share an amino-terminal phosphotyrosine-binding (PTB) domain, a central proline/glycine-rich region (CH1), and a carboxyl-terminal Src homology 2 (SH2) domain (6Nakamura T. Sanokawa R. Sasaki Y. Ayusawa D. Oishi M. Mori N. Oncogene. 1996; 13: 1111-1121PubMed Google Scholar, 7O'Bryan J.P. Songyang Z. Cantley L. Der C.J. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2729-2734Crossref PubMed Scopus (95) Google Scholar, 8Pelicci G. Lanfrancone L. Grignani F. McGlade J. Cavallo F. Forni G. Nicoletti I. Pawson T. Pelicci P.G. Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1131) Google Scholar, 9Pelicci G. Dente L., De Giuseppe A. Verducci-Galletti B. Giuli S. Mele S. Vetriani C. Giorgio M. Pandolfi P.P. Cesareni G. Pelicci P.G. Oncogene. 1996; 13: 633-641PubMed Google Scholar). ShcA exists in three isoforms in mammalian cells, p46, p52, and p66, which differ only in the extent of their amino-terminal sequence and are produced through alternative splicing and differential use of translation initiation sites (10Luzi L. Confalonieri S., Di Fiore P.P. Pelicci P.G. Curr. Opin. Genet. Dev. 2000; 10: 668-674Crossref PubMed Scopus (188) Google Scholar). ShcA has two modules of phosphotyrosine recognition with different specificities, an amino-terminal PTB domain and a carboxyl-terminal SH2 domain (11van der Geer P. Wiley S. Lai V.K. Olivier J.P. Gish G.D. Stephens R. Kaplan D. Shoelson S. Pawson T. Curr. Biol. 1995; 5: 404-412Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Thus, it can associate with tyrosine-phosphorylated proteins such as receptor tyrosine kinases and cytoplasmic proteins (e.g. SHIP2) (12Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Crossref PubMed Scopus (561) Google Scholar). ShcA has been found to be phosphorylated rapidly and efficiently by all tyrosine kinases tested to date (10Luzi L. Confalonieri S., Di Fiore P.P. Pelicci P.G. Curr. Opin. Genet. Dev. 2000; 10: 668-674Crossref PubMed Scopus (188) Google Scholar). These phosphorylation sites have been mapped to Tyr239, Tyr240, and Tyr317 in the CH1 domain (13Salcini A.E. McGlade J. Pelicci G. Nicoletti I. Pawson T. Pelicci P.G. Oncogene. 1994; 9: 2827-2836PubMed Google Scholar, 14Gotoh N. Tojo A. Shibuya M. EMBO J. 1996; 15: 6197-6204Crossref PubMed Scopus (115) Google Scholar, 15van der Geer P. Wiley S. Gish G.D. Pawson T. Curr. Biol. 1996; 6: 1435-1444Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). These phosphorylated tyrosines then serve as binding sites for the SH2 domain of adapter protein Grb2 (15van der Geer P. Wiley S. Gish G.D. Pawson T. Curr. Biol. 1996; 6: 1435-1444Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 16Rozakis-Adcock M. McGlade J. Mbamalu G. Pelicci G. Daly R., Li, W. Batzer A. Thomas S. Brugge J. Pelicci P.G. Schlessinger J. Pawson T. Nature. 1992; 360: 689-692Crossref PubMed Scopus (825) Google Scholar, 17Songyang Z. Shoelson S.E. McGlade J. Olivier P. Pawson T. Bustelo X.R. Barbacid M. Sabe H. Hanafusa H., Yi, T. et al.Mol. Cell. Biol. 1994; 14: 2777-2785Crossref PubMed Scopus (829) Google Scholar), which is constitutively associated with SOS, a ubiquitously expressed Ras guanine nucleotide exchange factor. This binding leads to recruitment of SOS to the plasma membrane, an event considered sufficient to induce Ras activation. Consistent with this model, overexpression of ShcA can transform mouse fibroblasts and induce differentiation of PC12 cells (8Pelicci G. Lanfrancone L. Grignani F. McGlade J. Cavallo F. Forni G. Nicoletti I. Pawson T. Pelicci P.G. Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1131) Google Scholar). In addition to tyrosine phosphorylation, p66Shc can also be phosphorylated at serine/threonine residues in response to epidermal growth factor (EGF) (18Okada S. Kao A.W. Ceresa B.P. Blaikie P. Margolis B. Pessin J.E. J. Biol. Chem. 1997; 272: 28042-28049Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), 12-O-tetradecanoylphorbol-13-acetate (TPA) (19El-Shemerly M.Y. Besser D. Nagasawa M. Nagamine Y. J. Biol. Chem. 1997; 272: 30599-30602Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), UV stress (20Migliaccio E. Giorgio M. Mele S. Pelicci G. Reboldi P. Pandolfi P.P. Lanfrancone L. Pelicci P.G. Nature. 1999; 402: 309-313Crossref PubMed Scopus (1460) Google Scholar), Taxol (21Yang C.P. Horwitz S.B. Cancer Res. 2000; 60: 5171-5178PubMed Google Scholar), and endothelin-1 (22Foschi M. Franchi F. Han J., La Villa G. Sorokin A. J. Biol. Chem. 2001; 276: 26640-26647Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). EGF-induced serine/threonine phosphorylation of p66Shc has been implicated in the negative regulation of the MAPK pathway (18Okada S. Kao A.W. Ceresa B.P. Blaikie P. Margolis B. Pessin J.E. J. Biol. Chem. 1997; 272: 28042-28049Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). In contrast, it has been suggested that the TPA-induced serine/threonine phosphorylation of p52Shc/p66Shc is involved in ERK activation because it leads to an increase in ShcA/Grb2 association without an increase in ShcA tyrosine phosphorylation (19El-Shemerly M.Y. Besser D. Nagasawa M. Nagamine Y. J. Biol. Chem. 1997; 272: 30599-30602Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Similarly, a protein kinase C (PKC)-dependent increase in Shc/Grb2 association after TPA treatment in MCF7 cells has been attributed to ERK activation (23Sato H. Ogata H. De Luca L.M. Oncogene. 2000; 19: 2904-2912Crossref PubMed Scopus (27) Google Scholar). Finally, Migliaccio et al. (20Migliaccio E. Giorgio M. Mele S. Pelicci G. Reboldi P. Pandolfi P.P. Lanfrancone L. Pelicci P.G. Nature. 1999; 402: 309-313Crossref PubMed Scopus (1460) Google Scholar) reported that serine phosphorylation of p66Shc regulates stress-induced apoptotic response and life span in mammals. It has been shown that p52Shc/p66Shc proteins are associated with the protein-tyrosine phosphatase PTP-PEST and that this association can be enhanced in HeLa cells by PKC activators like TPA, but not by EGF (24Habib T. Herrera R. Decker S.J. J. Biol. Chem. 1994; 269: 25243-25246Abstract Full Text PDF PubMed Google Scholar). The sites involved in the association of p52ShcA/p66ShcA and PTP-PEST were mapped to the PTB domain in ShcA and an NPLH sequence in the carboxyl terminus of PTP-PEST (25Charest A. Wagner J. Jacob S. McGlade C.J. Tremblay M.L. J. Biol. Chem. 1996; 271: 8424-8429Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). This was the first report showing phosphotyrosine-independent binding of the PTB domain of ShcA to a target protein. Murine PTP-PEST is a ubiquitously expressed cytosolic phosphatase of 112 kDa characterized by the presence of a so-called PEST sequence that is thought to confer protein stability (26Charest A. Wagner J. Shen S.H. Tremblay M.L. Biochem. J. 1995; 308: 425-432Crossref PubMed Scopus (62) Google Scholar). PTP-PEST is phosphorylated by PKC and protein kinase A at Ser39 and Ser435, and this serine phosphorylation down-regulates its activity (27Garton A.J. Tonks N.K. EMBO J. 1994; 13: 3763-3771Crossref PubMed Scopus (106) Google Scholar). Association with other proteins (28Mauro L.J. Dixon J.E. Trends Biochem. Sci. 1994; 19: 151-155Abstract Full Text PDF PubMed Scopus (180) Google Scholar) may also control the activity of protein-tyrosine phosphatases, and ShcA has been suggested to recruit PTP-PEST to its substrates for dephosphorylation (24Habib T. Herrera R. Decker S.J. J. Biol. Chem. 1994; 269: 25243-25246Abstract Full Text PDF PubMed Google Scholar). PTP-PEST has been shown recently to associate with and dephosphorylate ShcA in B cells, contributing to negative regulation of lymphocyte activation via inactivation of the Ras pathway (29Davidson D. Veillette A. EMBO J. 2001; 20: 3414-3426Crossref PubMed Scopus (100) Google Scholar). Activated insulin receptor phosphorylates various cellular substrates at tyrosine residues (30White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar). ShcA is one of these substrates and has been shown to play an important role in insulin-induced ERK activation (31Yamauchi K. Pessin J.E. J. Biol. Chem. 1994; 269: 31107-31114Abstract Full Text PDF PubMed Google Scholar, 32Sasaoka T. Draznin B. Leitner J.W. Langlois W.J. Olefsky J.M. J. Biol. Chem. 1994; 269: 10734-10738Abstract Full Text PDF PubMed Google Scholar, 33Ishihara H. Sasaoka T. Ishiki M. Takata Y. Imamura T. Usui I. Langlois W.J. Sawa T. Kobayashi M. J. Biol. Chem. 1997; 272: 9581-9586Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Tyrosine phosphorylation of ShcA at residue 317 has been shown to play an important role in signal transduction to MAPK by insulin in Rat1 fibroblasts expressing the insulin receptor (33Ishihara H. Sasaoka T. Ishiki M. Takata Y. Imamura T. Usui I. Langlois W.J. Sawa T. Kobayashi M. J. Biol. Chem. 1997; 272: 9581-9586Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). In this report, we have identified TPA- and growth factor-induced serine/threonine phosphorylation sites in ShcA. One of these phosphorylation sites has been characterized and shown to be involved in binding to the protein-tyrosine phosphatase PTP-PEST. Phosphorylation of this site was induced by overexpression of some constitutively active isoforms of PKC, but not by others. Furthermore, binding of PTP-PEST to Shc down-regulated insulin-induced ERK activation. We have shown also that TPA-induced ERK activation does not involve serine phosphorylation of Shc in mouse embryo fibroblasts. We thank Dr. Jerrold Olefsky for kindly providing HIRc-B cells, Dr. Tony Pawson for Shc−/− mouse embryo fibroblasts, Dr. Peter E. Shaw for GalSAP-1 and G5E4lux, and Drs. Michael D. Schaller and Michel L. Tremblay for anti-PTP-PEST antibodies. We are grateful to Jan Hofsteenge and Hoanh Tran for stimulating discussion during this work and Patrick King and Derek Brazil for critical reading of the manuscript. The Friedrich Miescher Institute is part of the Novartis Research Foundation.
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